Introduction

Skin cancer is the most common cancer in Americans, and its incidence is rising dramatically (1, 2). Although many environmental and genetic factors contribute to the development of skin cancer, the most important factor is chronic exposure of the skin to solar ultraviolet (UV) radiation (3, 4). The solar UV spectrum can be divided into 3 subtypes, each of which has distinct biologic effects: UVA (320–400 nm, long wave), UVB (280–320 nm, mid wave), and UVC (200–280 nm, short wave). Although UVC radiation is largely absorbed by stratospheric ozone, UVA and UVB reach the surface of the earth. UVA (90%–95% of the total solar UV radiation), is considered as the “aging ray” that can lead to benign tumor formation as well as malignant cancers through the generation of reactive oxygen species (3). However, UVB (5% of the total solar UV radiation) is mainly responsible for a variety of skin diseases, including nonmelanoma and melanoma skin cancers (1, 3, 5). It acts as a tumor initiator, tumor promoter and cocarcinogen by induction of oxidative stress, DNA damage, and immunosuppression (2, 3). UVB is recognized as a complete carcinogen with relevance to human skin cancer (2) and a potent inducer of mitogen-activated protein kinases (MAPK; refs. 1, 6, 7). MAPKs are serine–threonine kinases that control fundamental cellular processes such as growth, proliferation, differentiation, migration, and apoptosis (8–10). The mammalian MAPK family consists of extracellular signal–regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK; also known as stress-activated protein kinases or SAPK) and p38. Among the MAPK family, the ERKs cascade has been a focus of cancer chemoprevention because of its importance in carcinogenesis. Abnormalities in the ERKs pathway play a critical role in the development and progression of cancer and have been reported in approximately one third of all human cancers (8). Therefore, targeting UV-induced ERKs might be an effective strategy for preventing skin tumorigenesis.

Norathyriol has no cytotoxity in JB6 cells. A, chemical structure of norathyriol. B, cells were treated with norathyriol (0–50 μmol/L), or its vehicle, DMSO, as a negative control, in 5% FBS/MEM for 24 or 48 hours. Cell viability was determined by MTS assay and data are represented as means ± SE.

Materials and Methods

Chemicals

Eagle's minimum essential medium (EMEM) and basal medium Eagle were purchased from Invitrogen. FBS was purchased from Gemini Bio-products. The antibodies against phosphorylated ERKs (Tyr-202/Tyr-204), total ERKs, phosphorylated JNK (Thr-183/Tyr-185), total JNK, phosphorylated p90RSK (Thr-359/Ser-363), total p90RSK, phosphorylated p38 (Thr-180/Tyr-182), total p38, cyclin B1, phosphorylated Cdk1 (Tyr15), and total Cdk1 were purchased from Cell Signaling Biotechnology. The antibody against β-actin was purchased from Sigma-Aldrich. The Protein Assay Kit was from Bio-Rad, and the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit and the luciferase assay substrate were purchased from Promega. The active ERK1 and ERK2 kinases were obtained from Upstate Biotechnology. CNBr–Sepharose 4B and [γ-32P] ATP were purchased from GE Healthcare.

In silico virtual screening

Molecular docking-based virtual screening was used to screen the Chinese Medicine Library. The Glide software program (Schrödinger, Inc.) was used for virtual docking of compounds that used grid-based ligand docking with an energetics algorithm. Lipinski's rule of 5 was adopted for filtering out orally inactive molecules. For maximum diversity of chemicals, different tautomeric and ionization states were also generated. Thus the final library contains multiple states of some of the chemicals. The crystal structure of ERK2 with N, N-dimethyl-4-(4-phenyl-1H-pyrazol-3-yl)-1H-pyrrole-2-carboxamide (PDB ID 2OJG) bound in the ATP-binding site was used as a starting model for the virtual screening. After removing all the crystallographic water molecules, the protein structure was corrected by adding all the missing hydrogen atoms. Then the protein–ligand structure was subjected to energy minimization using the optimized potentials for liquid simulations-all atom (OPLS-AA) force field. A hierarchical filtering procedure on the basis of different levels of precision score function was used to identify potential inhibitors. Initial docking precision uses a high throughput virtual screening (HTVS) procedure, followed by a standard precision (SP) procedure, and finally the extra precision (XP) procedure. The binding affinity of the docked molecules can be considered as directly proportional to the docking score. The 25 molecules with a high XP score were selected as potential inhibitors of ERK2 with a potentially high affinity to bind with ERK2. The XP score of norathyriol was determined to be −9.6 kcal/mol.

Synthesis of norathyriol

The synthesis was carried out on the basis of a described procedure (21). Boron tribromide (1 mol/L in DCM, 100 mL, 2.5 eqv. per each OCH3 group) was added drop wise to a stirred suspension of 1, 3, 6, 7-tetramethoxyxanthone (3.16 g, 10 mmol in 25 mL dichloromethane at −78°C) over a period of 30 min under a nitrogen atmosphere. After the addition, the reaction mixture was slowly brought to room temperature and stirred at this temperature for 48 hours. After this time, the mixture was cooled to 0°C and excess boron tribromide was quenched by the slow addition of ice water. The resulting precipitate was filtered and dried under vacuum. The crude material was purified by column chromatography (20%–50% ethyl acetate in hexane) followed by recrystallization using methanol/water to yield 2.0 g (77%) of norathyriol. The compound was confirmed by 1H NMR and comparing it with an authentic commercially available sample. 1H NMR [400 MHz, dimethyl sulfoxide (DMSO)-d6]: δ 13.21 (s, 1H), 10.75 (br s, 1H), 7.36 (s, 1H), 6.84 (s, 1H), 6.31 (d, J = 1.8 Hz, 1H), 6.13 (d, J = 1.8 Hz, 1H); ESI MS 259 (M–H+).

Cytotoxicity assay

To estimate cytotoxicity, JB6 P+ cells were seeded (2 × 104 cells/well) in 96-well plates with 5% FBS/MEM at 37°C in a 5% CO2 incubator. Cells were fed with fresh medium after 4 hours and treated with norathyriol at the concentrations indicated (0, 1, 10, 25, or 50 μmol/L). After culturing for the indicated times, 20 μL of Cell Titer 96 AQueous One Solution were added to each well, and the cells were then incubated for 1 hour at 37°C in a 5% CO2 incubator. Absorbance was measured at 490 and 690 nm.

UVB irradiation

A UVB irradiation system was used to stimulate cells in serum-free media. The spectral peak from the UVB source (Bio-Link Crosslinker) was 312 nm.

Western blot analysis

After cells (1 × 106) were cultured in a 10-cm dish overnight, they were starved in serum-free medium for another 24 hours to eliminate the influence of FBS on the activation of MAPKs. The cells were then treated with norathyriol (0–25 μmol/L) for 2 hours before they were exposed to UVB (4 kJ/0.5 m2) and then harvested after 30 minutes. The harvested cells were disrupted, and the supernatant fractions were boiled for 5 minutes. The protein concentration was determined using a dye-binding protein assay kit (Bio-Rad) as described in the manufacturer's manual. Lysate proteins (30–50 μg) were subjected to 10% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (GE Healthcare). After blotting, the membrane was incubated with a specific primary antibody at 4°C overnight. Protein bands were visualized using a chemifluorescent detection kit (GE Healthcare) after hybridization with an AP-linked secondary antibody.

Luciferase assay to determine AP-1 or NF-κB transactivation

Confluent monolayers of JB6 P+ cells stably transfected with an AP-1 or NF-κB luciferase reporter plasmid were trypsinized, and 4 × 104 viable cells suspended in 1 mL of 5% FBS/MEM were added to each well of a 24-well plate. Plates were incubated overnight at 37°C in a humidified atmosphere of 5% CO2. Cells were starved in serum-free medium for another 24 hours. The cells were treated for 2 hours with norathyriol (0–25 μmol/L) and then exposed to UVB (4 kJ/m2) and harvested after 3 hours. Cells were then disrupted with 100 μL of lysis buffer (0.1 mol/L potassium phosphate pH 7.8, 1% Triton X-100, 1 mmol/L dithiothreitol (DTT), and 2 mmol/L EDTA), and luciferase activity was measured using a luminometer (Luminoskan Ascent, Thermo Electro).

In vitro ERK1 and ERK2 kinase assay

The recombinant nonphosphorylated GST-tagged RSK2 (residues 326–740; 500 ng) was used as a substrate in an in vitro kinase assay with 39.8 ng of active ERK1 or 10 ng of active ERK2 (Upstate Biotechnology). Reactions were carried out in 1× kinase buffer (25 mmol/L Tris-HCl pH 7.5, 5 mmol/L β-glycerophosphate, 2 mmol/L DTT, 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2) containing 50 μmol/L unlabeled ATP with or without 10 μCi of [γ-32P]ATP at 30°C for 30 minutes. Reactions were stopped and then proteins resolved by 10% SDS-PAGE and visualized by autoradiography.

ATP and norathyriol competition assay

ATP (1, 10, or 100 μmol/L) was mixed with active ERK2 (0.2 μg) to a final volume of 500 μL for 30 minutes. After this we added 100 μL norathyriol-Sepharose 4B (or Sepharose 4B for control) and incubated for 12 hours at 4°C. The samples were washed, and proteins were then detected by Western blot analysis.

Recombinant ERK2 expression, purification, and crystallization

Full length (residues 1–360) human ERK2 (NM_002745) was cloned into the pET-28a vector (EMD Chemicals) at the NdeI/HindIII restriction sites and expressed in BL21-CodonPlus (DE3)-RIPL Escherichia coli (Stratagene). The cells were induced with 0.25 mmol/L IPTG and grown for an additional 4 to 5 hours at 25°C. The frozen pellet was resuspended in washing buffer containing 20 mmol/L imidazole, 500 mmol/L NaCl, 50 mmol/L NaH2PO4 pH 8.0, and 10% glycerol. The cells were disrupted by French press. Soluble His-tagged ERK2 (about 90% of total protein) was loaded on Ni-NTA (Qiagen), washed thoroughly with washing buffer, eluted with 60, 100, and 200 mmol/L imidazole, 150 mmol/L NaCl, and 20 mmol/L Tris-HCl pH 8.0. All fractions were combined and loaded onto Econo-pack 10DG desalting columns (Bio-Rad) to exchange buffer for 150 mmol/L NaCl, 20 mmol/L Tris-HCl pH 8.0. To remove the His-tag, the protein was incubated with thrombin (EMD Chemicals) at 0.25 U thrombin/mg protein for 2 to 2.5 hours at room temperature. Untagged ERK2 was purified on a Superdex 200 10/30 GL FPLC column, pooled and concentrated to 10 to 15 mg/mL. Diffraction quality crystals were produced by the sitting drops vapor diffusion technique at 20°C. The protein was diluted with 10 mmol/L Tris-HCl pH 8.0 to 7 mg/mL, and 10 mmol/L β-mercaptoethanol was added prior to crystallization. The protein was mixed with an equal volume of precipitant comprising 1.1 to 1.3 mol/L ammonium sulfate, 2% PEG 500 MME, 0.1 mol/L HEPES-NaOH pH 7.5. The crystals appeared in 2 to 4 days. To produce the ERK2/norathyriol complex, the crystals of apo ERK2 were soaked overnight with the solution containing 2.5 mmol/L norathyriol. The compound from the stock solution (50 mmol/L in 100% DMSO) was added to a 2-fold diluted well solution (0.5–0.7 mol/L ammonium sulfate, 2% PEG 500 MME, 0.1 M HEPES-NaOH pH 7.5) at a final 5 mmol/L concentration. That solution was added to the preexisting drop with the crystals at a 1:1 (v:v) ratio. After soaking, the crystals were cryo-protected in buffer comprising 1.6 mol/L ammonium sulfate, 2% PEG 500 MME, 0.1 mol/L HEPES (pH 7.5), and 20% xylitol. They were then flash-cooled in liquid nitrogen. The His-tagged ERK2 mutant, Q105A, was purified by a similar procedure.

ERK2/norathyriol cocrystal structure determination

ERK2 crystals belong to the primitive monoclinic lattice P21 with 1 molecule in the asymmetric unit cell. The high-resolution data sets were collected at the Advanced Photon Source (APS) beamline 24ID using 30 × 50 micron beam. The crystal to Quantum 315 CCD detector distance was 220 mm and the crystals were rotated around the spindle axis with images collected over 180° to a resolution of 1.6 Å. Data were integrated and scaled using the HKL2000 package (22). Integrated intensities from 3 different crystals were scaled together to increase redundancy and improve overall statistics. The real resolution of the data, used for structure refinement, was estimated taking into consideration the completeness of the last resolution shell, I/σ ratio and R-merge values. The atomic coordinates of the refined ERK2 structure (PDB code 1TVO) were used for the initial crystallographic phasing by molecular replacement. All calculations were carried out using PHENIX (23). With the model given by molecular replacement, a rigid body refinement was carried out at 3.5 Å resolution. All data with a high-resolution limit of 1.6 Å were used for structure refinement with riding hydrogens. Once a satisfactory description of the protein electron density was complete, water molecules were added, followed by the modeling of norathyriol. A few cycles of slow-cooling annealing (2,500 → 100 K), positional, restrained isotropic temperature factor and TLS refinements were followed by visual inspection of the electron density maps, including omit maps, coupled with manual model building (when necessary) using the graphics program COOT (24). The refined electron density clearly matched the amino acid sequence of ERK2 with the exception of the N- and C-terminus (residues 3–17 and 358–361) and a weak electron density was observed for residues 23–34. Strong stereochemical restraints were imposed during the crystallographic refinement and the final ERK2 structure possessed a very good stereochemistry with a root mean square deviation (r.m.s.d.) of ∼0.013 Å for bond lengths and ∼1.4° for angles. The quality of the stereochemistry of the final protein structure was assessed with the PROCHECK package (25). The Ramachandran plot showed no residues in disallowed regions (data not shown). As a better guide to the quality of the structure, the values of the free R-factor were monitored during the course of the crystallographic refinement. The final value of free R-factors did not exceed the overall R-factor by more than 3%. The refined coordinates of ERK2/norathyriol complex structure have been deposited into the Protein Data Bank with PBD ID 3SA0.

Cell growth and death assays

JB6 P+ cells were seeded (8 × 104 cells/well) in 6-well plates with 5% FBS/MEM at 37°C in a 5% CO2 incubator overnight and then starved in a serum-free medium for 24 hours. They were then fed with fresh medium and treated with different doses of norathyriol (0, 1, 10, or 25 μmol/L). After 24 or 72 hours of treatment, total cells were collected by brief trypsinization, and washed with PBS. Total cell number was determined by counting each sample in duplicate using a hemocytometer under an inverted microscope. Cell viability was determined using the trypan blue exclusion method. The data shown in this study are the mean of 3 independent experiments.

Cell-cycle assay

JB6 P+ cells were seeded (2 × 105 cells/well) in 60 mm dishes with 5% FBS/MEM at 37°C in a 5% CO2 incubator overnight. Then cells were starved in a serum-free medium for 24 hours followed by treatment with norathyriol (0, 1, 10, or 25 μmol/L) for 12 or 24 hours in MEM containing 5% FBS. The cells were trypsinized, washed twice with cold PBS, and fixed with ice-cold 70% ethanol at −20°C overnight. Cells were then washed twice with PBS, incubated with 20 mg/mL RNase A and 200 mg/mL propidium iodide in PBS at room temperature for 30 minutes in the dark, and subjected to flow cytometry using the FACSCalibur flow cytometer. Data were analyzed using ModFit LT (Verity Software House, Inc.).

Mouse skin tumorigenesis study

Female SKH-1 hairless mice were purchased from the National Cancer Institute (NIH) and were maintained under “specific pathogen-free” conditions according to guidelines established by Research Animal Resources, University of Minnesota. The skin carcinogenesis experiments were conducted using mice at 5 to 6 weeks of age with a mean body weight of 25 g. Skin carcinogenesis in mice was induced using a solar UV irradiation system. The solar UV radiation source (Q-Lab Corporation) emitted at wavelengths of 295 to 365 nm and the peak emission was 340 nm. SKH-1 mice were divided into 4 groups of 10 animals each. In the control group, the dorsal skin was topically treated with 200 μL acetone only. In the solar UV–treated group, the dorsal skin was topically treated with 200 μL acetone 1 hour before UVB. The mice in groups 3 and 4 received topical application of norathyriol (0.5 or 1 mg, respectively) in 200 μL acetone 1 hour before solar UV irradiation. At week 1, the solar UV dose was 30 kJ/m2 UVA/1.8 kJ/m2 UVB given twice/week. The dose of solar UV was progressively increased (10% each week). At week 6, the dose was 48 kJ/m2 UVA/2.9 kJ/m2 UVB and this dose was maintained for weeks 6 to 15. The tumor was defined as an outgrowth of >1 mm in diameter that persisted for 2 weeks or more. Tumor numbers and volume were recorded every week until the end of the experiment. One-half of the samples were immediately fixed in 10% neutral-buffered formalin and processed for hematoxylin and Eosin (H&E) staining and immunostaining. The other samples were frozen and used for Western blot analysis.

Statistical analysis

Results

Hyperactivation of ERKs results in unregulated cell proliferation in several human cancers including skin cancer (26, 27), suggesting that inhibition of ERKs represents a potential approach for the prevention of cancer. Using in silico virtual screening, we found that norathyriol (Fig. 1A) might be a potential ERK2 inhibitor. First, we examined the cytotoxicity of this compound. The varying concentrations (0–50 μmol/L) of norathyriol had minimal effect on the viability of JB6 P+ cells harvested at 24 or 48 hours (Fig. 1B). We also measured the effect of norathyriol on UVB-induced MAPKs phosphorylation in JB6 P+ cells. Results showed that norathyriol inhibited UVB-induced phosphorylation of ERKs and RSK, but had no obvious effect on JNKs or p38 (Fig. 2A). The transcription factor activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB) are activated through the MAPKs pathway upon stimulation with UV (1, 28). To detect the transactivation of AP-1 and NF-κB, we exposed JB6 P+ cells stably transfected with an AP-1 or NF-κB luciferase reporter plasmid to norathyriol and this compound suppressed UVB-induced transactivation of AP-1 and NF-κB in a dose-dependent manner (Fig. 2B).

Norathyriol inhibits UVB-induced AP-1 and NF-κB transactivation in JB6 cells through inhibition of the ERKs signaling pathway. A, norathyriol inhibits UVB-induced phosphorylation of ERKs and p90RSK. Cells were treated with norathyriol at the indicated concentrations (0–25 μmol/L) for 2 hours, and then exposed to UVB (4 kJ/0.5 m2) and harvested after 30 minutes. The levels of phosphorylated and total ERKs, p90RSK, JNKs, and p38 proteins were determined by Western blot analysis. B, for the luciferase assay, JB6 cells stably transfected with an AP-1 or NF-κB luciferase reporter plasmid were cultured. Cells were starved in serum-free medium for 24 hours, and then treated with norathyriol (0–25 μmol/L), or its vehicle, DMSO (negative control), in serum-free medium for 2 hours. Cells were then exposed to UVB (4 kJ/m2) and harvested 3 hours later. Luciferase activity was measured and AP-1 or NF-κB activity is expressed relative to control cells without UVB treatment. Data are represented as means ± SE. The asterisk (*) indicates a significant difference (P < 0.05) between groups treated with UVB and norathyriol and the group treated with UVB alone.

ERKs are potential targets of norathyriol

We focused on ERKs signaling and determined the effects of norathyriol on the kinase activity of ERK1 and ERK2. Kinase assay data revealed that norathyriol (10 μM) strongly suppressed ERK1 and 2 activities in vitro (Fig. 3A). A direct interaction of norathyriol with ERK1 and 2 was shown by an in vitro pull-down assay (Fig. 3B). To examine the means by which norathyriol binds directly with ERK2, we carried out an ATP competition assay. The results indicated that norathyriol inhibits ERK2 activity competitively with ATP (Fig. 3C).

Norathyriol inhibits ERK1 and 2 kinase activities and directly binds with ERK1 or 2 in an ATP-competitive manner. A, norathyriol inhibits ERK1 or 2 kinase activity. An in vitro ERK1 or 2 kinase assay was carried out as described in Materials and Methods. A GST-RSK2 fusion protein was used in an in vitro kinase assay with active ERK1 or 2 and results were visualized by autoradiography. Coomassie blue staining of the GST fusion protein served as a loading control. Left, lane 1, positive control, which indicates that active ERK2 phosphorylates the GST RSK2 fusion protein; lanes 2 and 3, positive controls and indicate that increasing amounts of CAY10561 (commercially available ERK2 inhibitor) suppresses ERK2 kinase activity; lanes 4–6, increasing amounts of norathyriol suppress ERK2 kinase activity. Right, lane 1, positive control, which indicates that active ERK1 phosphorylates the GST-RSK2 fusion protein; lanes 2 and 3, increasing amounts of norathyriol suppress ERK1 kinase activity. B, norathyriol directly binds with ERK1 or 2. Binding of norathyriol with ERK1 or 2 was confirmed by immunoblotting using an antibody against ERK1 or 2. Lane 1 (input control), ERK1, or ERK2 protein standard; lane 2 (control), Sepharose 4B was used to pull down ERK1 or ERK2; and lane 3, ERK1 or ERK2 was pulled down using norathyriol-conjugated Sepharose 4B beads as described in Materials and Methods. C, norathyriol binds to ERK2 competitively with ATP. Active ERK2 (0.2 μg) was incubated with ATP at the indicated concentrations (0, 1, 10, 100 μmol/L) and 100 μL of norathyriol-Sepharose 4B or 100 μL of Sepharose 4B (as a negative control) beads in a reaction buffer to a final volume of 500 μL. The pulled down proteins were detected by Western blot as described in Materials and Methods. Lane 1 (input control), ERK2 protein standard; lane 2, negative control, indicating that ERK2 does not bind with Sepharose 4B; lane3, positive control, indicating that ERK2 binds with norathyriol-Sepharose 4B beads; lanes 4–6, increasing amounts of ATP inhibits norathyriol binding with ERK2.

To confirm further that ERKs are a molecular target for norathyriol, we initiated a crystallographic study. Human ERK2 was cloned and purified from E. coli, and successfully crystallized. To obtain the ERK2/norathyriol complex, the crystals were soaked with a solution containing norathyriol. The 3D structure of ERK2 in complex with norathyriol was refined to a resolution of 1.6 Å. The data collection and refinement statistics are presented in Supplementary Table S1. Norathyriol was found in the ATP-binding site, between the 2 kinase lobes (Fig. 4A and B). The xanthone moiety is located below the phosphate-binding loop and occupies a position similar to that of the adenine ring of ATP. Three hydrogen bonds are formed between norathyriol and the hinge loop of ERK2 that involves the side-chain of Gln105 and the backbone chain of Asp106 and Met108 (Fig. 4B and C). The side chain carboxyl of Gln105, the identified gatekeeper residue (29), formed a hydrogen bond with the 7-OH group of norathyriol. The backbones of Asp106 and Met108 form hydrogen bonds with the 6-OH group of norathyriol. A recombinant ERK2 mutant, Q105A, showed decreased binding to the Sepharose 4B beads conjugated with norathyriol (Fig. 4D). The Ile31, Val39, and Ala52 residues from the N-lobe contribute to the hydrophobic interactions with the xanthone moiety of norathyriol. The carbonyl oxygen of norathyriol orients toward the conserved Lys52 from the β3-strand, but forms no contacts with that residue (2.91 Å). Together, the hydrogen bonds and hydrophobic interactions stabilize norathyriol in the ATP-binding pocket.

The crystal structure of the ERK2/norathyriol complex. A, overall ribbon presentation showing norathyriol bound to the hinge loop, a cross-over connection between the N- and the C-lobes, in the ATP-binding pocket. The α-helices are shown in magenta, and the β-strands are shown in cyan. The norathyriol molecule in stick presentation is shown in green. B, close-up view of the active site showing the hydrogen bonding between norathyriol and the residues in the hinge loop. The Gln105, Asp106, and Met108 residues each form a hydrogen bond at 2.86 Å, 2.43 Å, and 2.86 Å, respectively. C, the 2|Fo| − |Fc| electron density map countered at 1.1σ. The hinge loop, including the residues Gln105, Asp106, and Met108, are shown in sticks presentation. D, comparison of norathyriol binding with a wild-type (WT) recombinant ERK2 or an ERK2 Q105A mutant. Proteins were incubated with norathyriol-conjugated Sepharose 4B beads, and analyzed by Western blot with an ERKs antibody.

Early studies showing a link between ERKs signaling and cell-cycle machinery included the demonstration that blockade of thrombin-induced growth of Chinese hamster lung fibroblasts correlated with suppression of ERKs activation (30). Similarly, dominant-negative forms of ERKs inhibited growth of NIH3T3 fibroblasts (31). Because norathyriol attenuated ERKs signaling, we assessed the effect of norathyriol on JB6 P+ cell growth, cell death and cell cycle. Cells were treated with different concentrations of the agent (1, 10, or 25 μmol/L, final concentration in medium) dissolved in DMSO (vehicle) for 24 or 72 hours. At the end of each treatment time, determination of total cell number as well as number of dead cells showed that norathyriol inhibits cell growth in a dose- as well as time-dependent manner but does not cause cell death (Fig. 5A and B). The middle dose of norathyriol (10 μmol/L) showed 29% and 52% decreases in total cell number after 24 and 72 hours of treatment, respectively (Fig. 5A). The high dose (25 μmol/L) decreased cell number by 45% and 68% after 24 and 72 hours of treatment, respectively (Fig. 5A). Using the trypan blue dye exclusion method, we observed that the decrease in cell number caused by norathyriol was not accompanied by an increase in cell death (Fig. 5B). These data suggested that a decrease in cell number was not attributable to a cell-death-inducing effect of norathyriol. We investigated the effect of norathyriol on cell-cycle progression to determine whether the inhibitory effect on proliferation is caused by modulation of cell-cycle progression. After treatment with norathyriol, cells were stained with propidium iodide and analyzed by flow cytometry. Cell-cycle distribution analysis showed that the high dose of norathyriol for 12 or 24 hours resulted in an increase in the number of cells in G2–M phase (Fig. 5C). The G2–M phase of the cell-cycle is reportedly controlled primarily by cyclin B1 and its associated catalytically active partner Cdk1 (32). Therefore, we tested the effect of norathyriol on cyclin B1 and Cdk1 expression and Western blot data showed increases in cyclin B1 and phosphorylated Cdk1 protein level after treatment for 72 hours. These results suggested norathyriol inhibits cell growth by inducing cell-cycle G2–M arrest.

Norathyriol inhibits JB6 cell growth by inducing G2–M arrest. Cells were starved in serum-free medium for 24 hours and then treated with norathyriol (0–25 μmol/L), or its vehicle, DMSO (control), in 5% FBS/MEM for 24 or 72 hours. At the end of each treatment time, both floating and attached cells were collected and processed for (A) determination of total cell number and (B) number of dead cells. Data are represented as means ± SE. The asterisk (*) indicates a significant difference (P < 0.05) between groups treated with norathyriol and the group treated with DMSO. C, cells were starved in serum-free medium for 24 hours and then treated with norathyriol (0–25 μmol/L), or DMSO (control), for 12 or 24 hours. Cell-cycle analysis was carried out by flow cytometry. Data are represented as means ± SE. The asterisk (*) indicates a significant difference (P < 0.05) between groups treated with norathyriol and the group treated with DMSO. D, For Western blot analysis, cells were treated with norathyriol at the indicated concentrations for 72 hours.

To study the anti-tumorigenic activity of norathyriol in vivo, we evaluated the effect of norathyriol in a solar UV–induced mouse skin tumorigenesis model. Photographic data showed that norathyriol inhibited skin cancer development in mice treated with solar UV compared with mice treated only with solar UV (Fig. 6A). Topically applied norathyriol (0.5 or 1 mg) on mouse skin resulted in a significant inhibition of average tumor number per mouse (P < 0.05; Fig. 6B). The volume of tumors developed in solar UV–treated mouse skin was also significantly attenuated by norathyriol treatment (P < 0.05; Fig. 6C). At the end of the study, skin and tumor samples were processed for H&E staining. After treatment with solar UV, epidermal thickness increased by edema and epithelial cell proliferation, whereas norathyriol significantly inhibited epidermal thickness and inflammation (Fig. 6D). Immunostaining throughout the skin showed that chronic exposure to solar UV strongly increased phosphorylated and total ERKs expression compared with mice exposed to solar UV but treated only with acetone. However, treatment of skin with norathyriol resulted in a downregulation of phosphorylated and total ERKs levels (Fig. 6E). Overall, these results indicate that norathyriol might serve as an effective chemopreventive agent against solar UV–mediated skin cancer.

Norathyriol inhibits solar UV–induced skin tumorigenesis in SKH-1 hairless mice. Mice were divided into 4 groups: no solar UV irradiation (control); solar UV (SUV) treated; and treated with 0.5 or 1 mg of norathyriol in 200 μL of acetone 1 hour before SUV treatment. The compound was applied topically to the dorsal surface of each mouse before solar UV exposure as detailed in Materials and Methods. A, external appearance of tumors. B, average tumor number per mouse. Data are represented as means ± SE. The asterisk (*) indicates a significant difference (P < 0.05) between the groups treated with norathyriol + SUV and the group treated with acetone + SUV. C, tumor volume was calculated using the formula: tumor volume (mm3) = (length × width × height × 0.52). Data are represented as means ± SE. The asterisk (*) indicates a significant difference (P < 0.05) between the SUV groups treated with norathyriol and the group treated with acetone. At the end of the study, skin and tumor samples were fixed in 10% neutral-buffered formalin and processed for (D) H&E staining and (E) immunostaining with specific primary antibodies to detect phosphorylated ERKs (Tyr-202/Tyr-204) and total ERKs.

Discussion

Epidemiological, clinical, and laboratory studies showed that chronic solar UV radiation exposure-induced skin cancer is caused by the excessive induction of inflammation, oxidative stress, and DNA damage (3). The use of chemopreventive agents, especially naturally occurring plant products, to inhibit these events in UV-exposed skin is gaining more and more attention. A variety of phytochemicals have been reported to possess substantial skin photoprotective effects (2, 3, 5). Mangiferin is a xanthone (2-b-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one) widely distributed in mango (11). Norathyriol is a main metabolite of mangiferin in vivo, derived from a deglycosylation process (33, 34). Norathyriol possesses antioxidant, anti-inflammatory and antitumor effects. Recent studies showed that norathyriol is more active than mangiferin (11, 14). In the present study, we have shown a chemopreventive effect of norathyriol against UV-induced skin cancer development and identified a molecular mechanism(s) and possible protein target(s).

Cellular homeostasis, the equilibrium between cell proliferation and cell death is controlled by cell-cycle progression and apoptosis induction (35). Unchecked proliferative potential involving deregulation of cell-cycle progression is generally described as a central process in the development of cancer (36). We examined the effects of norathyriol on mouse epidermal JB6 P+ cell proliferation, cell death and cell-cycle. The data showed that the inhibition of proliferation induced by norathyriol was associated with a G2–M cell-cycle arrest. ERK1 and ERK2 are reported to be involved in regulating the cell-cycle in mammalian cells (10). An early study showed that in the G1 phase and around the M phase, ERK1 and ERK2 are activated in CHO cells (37). Recent data suggested that ERK1 and ERK2 are required for epidermal G2–M progression (38). Growth inhibition caused by ERK1/2 loss is rescued by reintroducing ERK2, but not by activating ERKs effectors that promote G1 cell-cycle progression (38). Some natural compounds, such as oridonin (39) and Gleditsia sinensis thorn extract, reportedly induce G2–M arrest through ERK signaling (40). Our data showed that induction of cell-cycle arrest by norathyriol might be mediated through ERKs signaling because norathyriol strongly suppresses ERK1 and 2 phosphorylation and kinase activities. Animal study results revealed that treatment with norathyriol inhibits solar UV–induced carcinogenesis by blocking ERKs activation.

AP-1 (activator protein-1) is a transcription factor that is extremely important in the processes of cell proliferation, cell differentiation, inflammation, cell survival, cell transformation, as well as playing a major role in tumorigenesis (2, 5, 41). ERKs play a critical role in the transcriptional activity of AP-1. AP-1 and ERKs are key molecules activated after UVB exposure. Our results showed that norathyriol inhibits AP-1 transcriptional activation through suppression of the ERKs pathway. Norathyriol inhibited UVB-induced phosphorylation of ERKs and p90RSK. Using in silico virtual screening, we found that the molecular target of norathyriol might be ERKs. The results showed that norathyriol strongly inhibited ERKs kinase activity and that the inhibition resulted from ATP-competitive binding of norathyriol with ERKs.

The ERK2/norathyriol cocrystal structure presented herein first showed that ERKs might bind with xanthone-based compounds. Norathyriol was found to occupy the ATP-binding site with the xanthone moiety acting as an adenine mimetic and anchoring the compound to the hinge region by hydrogen bonds. The involvement of amino acids, Gln105, Asp106, and Met 108 residing at the hinge loop, in the interaction with inhibitors was previously observed in the ERK2/pyrazolo[3,4-c]pyridazine derivative complex structure (42) and in the ERK2/FR 180204 complex structure (26). The Met108 residue was also involved in hydrogen bonding with olomoucine (43). The hydrogen bonding between inhibitor and the backbone nitrogen of methionine in the hinge loop (Met108 in ERK2) is conserved in all ATP-competitive kinase inhibitors (44). Overall, the ERK2/norathyriol complex structure is similar to the previously reported ERK2 structures complexed with different compounds (26).

In summary, our results clearly showed that topical application of norathyriol markedly suppressed the formation of skin cancer in SKH-1 hairless mice exposed to solar UV. The inhibition by norathyriol occurs mainly through the suppression of proliferation by inducing G2–M arrest and attenuation of AP-1 activity that in turn downregulates ERKs expression. Norathyriol might be a new chemopreventive agent that is highly effective against UV-induced skin cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This study was supported by award RR-15301 from the National Center for Research Resources at the NIH. Use of the APS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under contract no. W-31-109-ENG-38; The Hormel Foundation, and NIH grants R37 CA081064, CA027502, CA120388, and ES016548.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

The crystallography work is based upon research conducted at the Northeastern Collaborative Access Team beamlines 24ID of the Advanced Photon Source (APS).

Footnotes

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).